Based on current data from the United Network for Organ Sharing (UNOS), cardiac allograft vasculopathy (CAV) is the leading cause of death after the first year of transplantation and accounts for 25% of all deaths annually after the third year. Initial studies from our laboratory have demonstrated that the expression of inducible nitric oxide synthase (iNOS; NOS2) can suppress the development of neointimal hyperplasia and prevent the development of CAV. The proposal described herein will test the hypothesis that viral vectors capable of transferring the human iNOS gene can suppress the development of CAV without causing undue toxicity to cardiac myocytes. Furthermore, this proposal will serve as the pre-clinical basis for near-term clinical trials for the future prevention of CAV in humans. In order to serve these ends, we will carry out the following Specific Aims: 1) to determine the efficacy of several viral vectors (adenoviral, adeno-associated virus, and lentivirus) containing the human iNOS gene (Ad-iNOS, AAV-iNOS, Lt-iNOS) and their respective control vectors for suppression of cardiac transplant arteriosclerosis in the rat model; 2) to determine toxicity of Ad-iNOS, AAV-iNOS, and Lt- iNOS and their respective control vectors in rat models; 3) to determine efficacy and toxicity of the ideal viral vector (Ad-iNOS, AAV-iNOS, or Lt-iNOS) and its respective control vector as determined in Specific Aim #2, in a porcine model of chronic rejection and 4) to determine the toxicity in human hearts that are being supported with ventricular assist devices. In this proposal we plan to address several key questions including 1) what effect will chronic iNOS expression have on myocardial contractility; 2) will chronic iNOS expression induce cardiac myocyte apoptosis; 3) will iNOS expression induce acute cellular rejection; 4) what effect will iNOS expression during an episode of acute cellular rejection have on global cardiac function and 5) can iNOS be delivered in a safe and efficacious manner such that CAV can be prevented in a large animal model of chronic rejection? We plan to address these questions using both in vitro and in vivo models. Upon completion of the specific alms outlined whin this proposal our goal will be utilize the data obtained from these experiment to serve as a guideline by which to proceed with a safe and efficacious iNOS-based gene therapy trial for the therapeutic prevention of CAV.